PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED FOR TRANSLATION IN ITS CODING REGIONS

ABSTRACT

The present invention relates to a pharmaceutical composition comprising a modified mRNA that is stabilised by sequence modifications and optimised for translation. The pharmaceutical composition according to the invention is particularly well suited for use as an inoculating agent, as well as a therapeutic agent for tissue regeneration. In addition, a process is described for determining sequence modifications that promote stabilisation and translational efficiency of modified mRNA of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/487,425, filed Sep. 16, 2014, which is a divisional of U.S.application Ser. No. 10/729,830, filed Dec. 5, 2003, which is aContinuation-In-Part of PCT Application No. PCT/EP02/06180 filed Jun. 5,2002, which in turn, claims priority from German Application No. DE 10127 283.9, filed Jun. 5, 2001. Applicants claim the benefits of 35 U.S.C.§120 as to the U.S. applications and PCT application and priority under35 U.S.C. §119 as to the said German application, and the disclosures ofall of the above-referenced applications are incorporated herein intheir entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a pharmaceutical composition containingan mRNA that is stabilised by sequence modifications in the translatedregion and is optimised for translation. The pharmaceutical compositionaccording to the invention is suitable in particular as an inoculatingagent and also as a therapeutic agent for tissue regeneration.Furthermore, a process for determining sequence modifications thatstabilise mRNA and optimise mRNA translation is disclosed.

2. Description of the Prior Art

Gene therapy and genetic vaccination are tools of molecular medicinewhose use in the treatment and prevention of diseases has considerablepotential. Both of these approaches are based on the incorporation ofnucleic acids into a patient's cells or tissue as well as on thesubsequent processing of the information encoded by the incorporatednucleic acids, i.e. the expression of the desired polypeptides.

Conventional procedures involved in previous applications of genetherapy and genetic vaccination involved the use of DNA in order toincorporate the required genetic information into a cell. In thisconnection various processes for the incorporation of DNA into cellshave been developed, such as for example calcium phosphate transfection,polyprene transfection, protoplast fusion, electroporation,microinjection and lipofection, in which connection lipofection inparticular has proved to be a suitable process.

A further process that has been suggested in particular for the case ofgenetic vaccination involves the use of DNA viruses as DNA vehicles.Because such viruses are infectious, a very high transfection rate canbe achieved when using DNA viruses as vehicles. The viruses used aregenetically altered so that no functional infectious particles areformed in the transfected cell. Despite this precautionary measure,however, the risk of uncontrolled propagation of the introducedtherapeutic gene as well as viral genes remains due to the possibilityof recombination events.

Normally DNA incorporated into a cell is integrated to a certain extentinto the genome of the transfected cell. On the one hand this phenomenoncan exert a desirable effect, since in this way a long-lasting action ofthe introduced DNA can be achieved. On the other hand the integrationinto the genome brings with it a significant risk for gene therapy. Suchintegration events may, for example, involve an insertion of theincorporated DNA into an intact gene, which produces a mutation thatinterferes with or completely ablates the function of the endogenousgene. As a result of such integration events, enzyme systems that areimportant for cellular viability may be switched off. Alternatively,there is also the risk of inducing transformation of the transfectedcell if the integration site occurs in a gene that is critical forregulating cell growth. Accordingly, when using DNA viruses astherapeutic agents and vaccines, a carcinogenic risk cannot be excluded.In this connection it should also be borne in mind that, in order toachieve effective expression of the genes incorporated into the cell,the corresponding DNA vehicles comprise a strong promoter, for examplethe viral CMV promoter. The integration of such promoters into thegenome of the treated cell may, however, lead to undesirable changes inthe regulation of the gene expression in the cell.

A further disadvantage of the use of DNA as a therapeutic agent orvaccine is the induction of pathogenic anti-DNA antibodies in thepatient, resulting in a potentially fatal immune response.

In contrast to DNA, the use of RNA as a therapeutic agent or vaccine isregarded as significantly safer. In particular, use of RNA is notassociated with a risk of stable integration into the genome of thetransfected cell. In addition, no viral sequences such as promoters arenecessary for effective transcription of RNA. Beyond this, RNA isdegraded rapidly in vivo. Indeed, the relatively short half-life of RNAin circulating blood, as compared to that of DNA, reduces the risksassociated with developing pathogenic anti-RNA antibodies. Indeed,anti-RNA antibodies have not been detected to date. For these reasonsRNA may be regarded as the molecule of choice for molecular medicinetherapeutic applications.

However, some basic problems still have to be solved before medicalapplications based on RNA expression systems can be widely employed. Oneof the problems in the use of RNA is the reliable, cell-specific andtissue-specific efficient transfer of the nucleic acid. Since RNA isnormally found to be very unstable in solution, up to now RNA could notbe used or used only very inefficiently as a therapeutic agent orinoculating agent in the conventional applications designed for DNA use.

Enzymes that break down RNA, so-called RNases (ribonucleases), areresponsible in part for the instability. Even minute contamination byribonucleases is sufficient to degrade down RNA completely in solution.Moreover, the natural decomposition of mRNA in the cytoplasm of cells isexquisitely regulated. Several mechanisms are known which contribute tothis regulation. The terminal structure of a functional mRNA, forexample, is of decisive importance. The so-called “cap structure” (amodified guanosine nucleotide) is located at the 5′ end and a sequenceof up to 200 adenosine nucleotides (the so-called poly-A tail) islocated at the 3′ end. The RNA is recognised as mRNA by virtue of thesestructures and these structures contribute to the regulatory machinerycontrolling mRNA degradation. In addition there are further mechanismsthat stabilise or destabilise RNA. Many of these mechanisims are stillunknown, although often an interaction between the RNA and proteinsappears to be important in this regard. For example, an mRNAsurveillance system has been described (Hellerin and Parker, Annu. Rev.Genet. 1999, 33: 229 to 260), in which incomplete or nonsense mRNA isrecognised by specific feedback protein interactions in the cytosol andis made accessible to decomposition. Exonucleases appear to contributein large measure to this process.

Certain measures have been proposed in the prior art to improve thestability of RNA and thereby enable its use as a therapeutic agent orRNA vaccine.

In EP-A-1083232 a process for the incorporation of RNA, in particularmRNA, into cells and organisms has been proposed in order to solve theaforementioned problem of the instability of RNA ex vivo. As describedtherein, the RNA is present in the form of a complex with a cationicpeptide or protein.

WO 99/14346 describes further processes for stabilising mRNA. Inparticular, modifications of the mRNA are proposed that stabilise themRNA species against decomposition by RNases. Such modifications mayinvolve stabilisation by sequence modifications, in particular reductionof the C content and/or U content by base elimination or basesubstitution. Alternatively, chemical modifications may be used, inparticular the use of nucleotide analogues, as well as 5′ and 3′blocking groups, an increased length of the poly-A tail as well as thecomplexing of the mRNA with stabilising agents, and combinations of theaforementioned measures.

In U.S. Pat. No. 5,580,859 and U.S. Pat. No. 6,214,804 mRNA vaccines andmRNA therapeutic agents are disclosed inter alia within the scope of“transient gene therapy” (TGT). Various measures are described thereinfor enhancing the translation efficiency and mRNA stability that relatein particular to the composition of the non-translated sequence regions.

Bieler and Wagner (in: Schleef (Ed.), Plasmids for Therapy andVaccination, Chapter 9, pp. 147 to 168, Wiley-VCH, Weinheim, 2001)report on the use of synthetic genes in combination with gene therapymethods employing DNA vaccines and lentiviral vectors. The constructionof a synthetic gag-gene derived from HIV-1 is described, in which thecodons have been modified with respect to the wild type sequence(alternative codon usage) in such a way as to correspond to frequentlyused codons found in highly expressed mammalian genes. In this way, inparticular, the A/T content compared to the wild type sequence wasreduced. Moreover, the authors found an increased rate of expression ofthe synthetic gag gene in transfected cells. Furthermore, increasedantibody formation against the gag protein was observed in miceimmunised with the synthetic DNA construct. An increase in cytokinerelease in vitro from transfected spleen cells of such mice was alsoobserved. Finally, an induction of a cytotoxic immune response in miceimmunised with the gag expression plasmid was also found. The authors ofthis article attribute the improved properties of their DNA vaccine to achange in the nucleocytoplasmic transport of the mRNA expressed by theDNA vaccine, which was due to the optimised codon usage. The authorsmaintain that the effect of the altered codon usage on the translationefficiency was only slight.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a new system for genetherapy and genetic vaccination that overcomes the disadvantagesassociated with the properties of DNA therapeutic agents and DNAvaccines and increases the effectiveness of therapeutic agents based onRNA species.

This object is achieved by the embodiments of the present inventioncharacterised in the claims.

In particular, a modified mRNA, as well as a pharmaceutical compositioncomprising at least one modified mRNA of the present invention and apharmaceutically compatible carrier and/or vehicle are provided. Themodified mRNA encodes at least one biologically active or antigenicpeptide or polypeptide, wherein the sequence of the mRNA comprises atleast one modification as set forth herein below as compared to the wildtype mRNA. Such modifications may be located in the region coding forthe at least one peptide or polypeptide, or in untranslated regions.

In one aspect, the G/C content of the region of the modified mRNA codingfor the peptide or polypeptide is increased relative to that of the G/Ccontent of the coding region of the wild type mRNA coding for thepeptide or polypeptide. The encoded amino acid sequence, however,remains unchanged compared to the wild type. (i.e. silent with respectto the encoded amino acid sequence).

This modification is based on the fact that, for efficient translationof an mRNA, the sequence of the region of the mRNA to be translated isessential. In this connection the composition and the sequence of thevarious nucleotides play an important role. In particular sequences withan increased G (guanosine)/C (cytosine) content are more stable thansequences with an increased A (adenosine)/U (uracil) content. Inaccordance with the invention, the codons are varied compared to thewild type mRNA, while maintaining the translated amino acid sequence, sothat they contain increased amounts of G/C nucleotides. Since severaldifferent codons can encode the same amino acid, due to degeneracy ofthe genetic code, the codons most favourable for the stability of themodified mRNA can be determined and incorporated (alternative codonusage).

Depending on the amino acid encoded by the modified mRNA, variouspossibilities for modifying the mRNA sequence compared to the wild typesequence are feasible. In the case of amino acids that are encoded bycodons that contain exclusively G or C nucleotides, no modification ofthe codon is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGCor CGG), Ala (GCC or GCG) and Gly (GGC or GGG) do not require anyalteration since no A or U is present.

In the following cases the codons that contain A and/or U nucleotidesare altered by substituting other codons that code for the same aminoacids, but do not contain A and/or U. Examples include: the codons forPro, which may be changed from CCU or CCA to CCC or CCG; the codons forArg, which may be changed from CGU or CGA or AGA or AGG to CGC or CGG;the codons for Ala, which may be changed from GCU or GCA to GCC or GCG;the codons for Gly, which may be changed from GGU or GGA to GGC or GGG.

In other cases, wherein A and/or U nucleotides may not be eliminatedfrom the codons, it is however possible to reduce the A and U content byusing codons that contain fewer A and/or U nucleotides. For example: thecodons for Phe, which may be changed from UUU to UUC; the codons forLeu, which may be changed from UUA, CUU or CUA to CUC or CUG; the codonsfor Ser, which may be changed from UCU or UCA or AGU to UCC, UCG or AGC;the codon for Tyr, which may be changed from UAU to UAC; the stop codonUAA, which may be changed to UAG or UGA; the codon for Cys, which may bechanged from UGU to UGC; the codon for His, which may be changed fromCAU to CAC; the codon for Gln, which may be changed from CAA to CAG; thecodons for Ile, which may be changed from AUU or AUA to AUC; the codonsfor Thr, which may be changed from ACU or ACA to ACC or ACG; the codonfor Asn, which may be changed from AAU to AAC; the codon for Lys, whichmay be changed from AAA to AAG; the codons for Val, which may be changedfrom GUU or GUA to GUC or GUG; the codon for Asp, which may be changedfrom GAU to GAC; the codon for Glu, which may be changed from GAA toGAG.

In the case of the codons for Met (AUG) and Trp (UGG) there is howeverno possibility of modifying the sequence.

The substitutions listed above may be used individually and in allpossible combinations in order to increase the G/C content of a modifiedmRNA compared to the original sequence. Thus, for example all codons forThr occurring in the original (wild type) sequence can be altered to ACC(or ACG). Preferably, however, combinations of the substitutionpossibilities given above are employed, for example: substitution of allcodons coding in the original sequence for Thr to ACC (or ACG) andsubstitution of all codons coding for Ser to UCC (or UCG or AGC);substitution of all codons coding in the original sequence for Ile toAUC and substitution of all codons coding for Lys to AAG andsubstitution of all codons coding originally for Tyr to UAC;substitution of all codons coding in the original sequence for Val toGUC (or GUG) and substitution of all codons coding for Glu to GAG andsubstitution of all codons coding for Ala to GCC (or GCG) andsubstitution of all codons coding for Arg to CGC (or CGG); substitutionof all codons coding in the original sequence for Val to GUC (or GUG)and substitution of all codons coding for Glu to GAG and substitution ofall codons coding for Ala to GCC (or GCG) and substitution of all codonscoding for Gly to GGC (or GGG) and substitution of all codons coding forAsn to AAC; substitution of all codons coding in the original sequencefor Val to GUC (or GUG) and substitution of all codons coding for Phe toUUC and substitution of all codons coding for Cys to UGC andsubstitution of all codons coding for Leu to CUG (or CUC) andsubstitution of all codons coding for Gln to CAG and substitution of allcodons encoding Pro to CCC (or CCG); etc.

Preferably the G/C content of the region of the modified mRNA coding forthe peptide or polypeptide is increased by at least 7%, more preferablyby at least 15%, and particularly preferably by at least 20% compared tothe G/C content of the coded region of the wild type mRNA encoding forthe polypeptide.

In this connection it is particularly preferred to maximise the G/Ccontent of the modified mRNA as compared to that of the wild typesequence. For some applications, it may be particularly advantageous tomaximise the G/C content of the modified mRNA in the region encoding theat least one peptide or polypeptide.

In accordance with the invention, a further modification of the mRNAcomprised in the pharmaceutical composition of the present invention isbased on an understanding that the translational efficiency is alsoaffected by the relative abundance of different tRNAs in various cells.A high frequency of so-called “rare” codons in an RNA sequence, whichare recognized by relatively rare tRNAs, tends to decrease thetranslational efficiency of the corresponding mRNA, whereas a highfrequency of codons recognized by relatively abundant tRNAs tends toenhance the translational efficiency of a corresponding mRNA.

Thus, according to the invention, the modified mRNA (which is containedin the pharmaceutical composition) comprises a region coding for thepeptide or polypeptide which is changed compared to the correspondingregion of the wild type mRNA so as to replace at least one codon of thewild type sequence that is recognized by a rare cellular tRNA with acodon recognized by an abundant cellular tRNA, wherein the abundant andrare cellular tRNAs recognize the same amino acid. In other words, thesubstituted codon in the modified mRNA, which is recognized by arelatively frequent tRNA, encodes the same amino acid as the wild type(unmodified) codon.

Through such modifications, the RNA sequences are modified so thatcodons are inserted/substituted that are recognized by abundantlyexpressed cellular tRNAs. Modifications directed to altering codon usagein a nucleic acid sequence to optimise expression levels of polypeptidesencoded therefrom are generally referred to in the art as “codonoptimisation”.

Those tRNAs which are abundant or rare in a particular cell are known toa person skilled in the art; see for example Akashi, Curr. Opin. Genet.Dev. 2001, 11(6): 660-666. Each organism has a preferred choice ofnucleotide or codon usage to encode any particular amino acid. Differentspecies vary in their codon preferences for translating mRNA intoprotein. The codon preferences of a particular species in which amodified mRNA of the present invention is to be expressed will,therefore, at least in part dictate the parameters of codon optimisationfor a nucleic acid sequence.

By means of this modification, according to the invention all codons ofthe wild type sequence that are recognized by a relatively rare tRNA ina cell may in each case be replaced by a codon that is recognized by arelatively abundant tRNA. As described herein, however, the codingsequence of the peptide or polypeptide is preserved. That is, arelatively abundant tRNA species, which replaces a relatively rare tRNAspecies in a modified mRNA of the invention, recognizes an amino acididentical to that recognized by the rare tRNA species.

According to the invention, it is particularly preferred to couple thesequential increase in the G/C fraction of a modified mRNA(particularly, for example, a maximally modified G/C content), with anincrease in the number of codons recognized by abundant tRNAs, whereinthe amino acid sequence of the peptide or polypeptide (one or more)encoded by the mRNA remains unaltered. This preferred embodimentprovides a particularly preferred mRNA species, possessing properties ofefficient translation and improved stability. Such preferred mRNAspecies are well suited, for example, for the pharmaceuticalcompositions of the present invention.

Sequences of eukaryotic mRNAs frequently include destabilising sequenceelements (DSE) to which signal proteins can bind and thereby regulatethe enzymatic degradation of the mRNA in vivo. Accordingly, for thefurther stabilisation of a modified mRNA of the invention, which may bea component of a pharmaceutical composition of the invention, one ormore changes may be made in the wild type mRNA sequence encoding the atleast one peptide or polypeptide, so as to reduce the number ofdestabilising sequence elements present. In accordance with theinvention, DSEs located anywhere in an mRNA, including the coding regionand in the non-translated regions (3′ and/or 5′ UTR), may be mutated orchanged to generate a modified mRNA having improved properties.

Such destabilising sequences are for example AU-rich sequences (“AURES”)that occur in 3′-UTR regions of a number of unstable mRNAs (Caput etal., Proc. Natl. Acad. Sci. USA 1986, 83: 1670-1674). The RNA moleculescontained in the pharmaceutical composition according to the inventionare therefore preferably altered as compared to the wild type mRNA so asto reduce the number of or eliminate these destabilising sequences. Suchan approach also applies to those sequence motifs recognised bypotential endonucleases. Such sequences include, for example, GAACAAG,which is found in the 3′UTR of the gene encoding the transferringreceptor (Binder et al., EMBO J. 1994, 13: 1969-1980). Sequence motifsrecognized by endonucleases are also preferably reduced in number oreliminated in the modified mRNA of the pharmaceutical compositionaccording to the invention.

Various methods are known to the person skilled in the art that aresuitable for the substitution of codons in the modified mRNA accordingto the invention. In the case of relatively short coding regions (thatcode for biologically active or antigenic peptides), the whole mRNA may,for example, be chemically synthesised using standard techniques.

Preferably, however, base substitutions are introduced using a DNAmatrix for the production of modified mRNA with the aid of techniquesroutinely employed in targeted mutagenesis; see Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 3^(rd) Edition, Cold Spring Harbor, N. Y., 2001.

In this method, a corresponding DNA molecule is therefore transcribed invitro for the production of the mRNA. This DNA matrix has a suitablepromoter, for example a T7 or SP6 promoter, for in vitro transcription,followed by the desired nucleotide sequence for the mRNA to be producedand a termination signal for the in vitro transcription. According tothe invention the DNA molecule that forms the matrix of the RNAconstruct to be produced is prepared as part of a plasmid replicable inbacteria, wherein the plasmid is replicated or amplified during thecourse of bacterial replication and subsequently isolated by standardtechniques. Plasmids suitable for use in the present invention include,but are not limited to pT7 Ts (GenBank Accession No. U26404; Lai et al.,Development 1995, 121: 2349-2360), the pGEM® series, for example pGEM®-1(GenBank Accession No. X65300; from Promega) and pSP64(GenBank-Accession No. X65327); see also Mezei and Storts, Purificationof PCR Products, in: Griffin and Griffin (Eds.), PCR Technology: CurrentInnovation, CRC Press, Boca Raton, Fla., 2001.

Thus, by using short synthetic DNA oligonucleotides that comprise shortsingle-strand transitions at the corresponding cleavage sites, or bymeans of genes produced by chemical synthesis, the desired nucleotidesequence can be cloned into a suitable plasmid by molecular biologymethods known to the person skilled in the art (see Maniatis et al.,above). The DNA molecule is then excised from the plasmid, in which itmay be present as a single copy or multiple copies, by digestion withrestriction endonucleases.

The modified mRNA that is contained in the pharmaceutical compositionaccording to the invention may furthermore have a 5′ cap structure (amodified guanosine nucleotide). Examples of suitable cap structuresinclude, but are not limited to m7G(5′)ppp (5′(A,G(5′)ppp(5′)A andG(5′)ppp(5′)G.

According to a further preferred embodiment of the present invention themodified mRNA comprises a poly-A tail of at least 50 nucleotides,preferably at least 70 nucleotides, more preferably at least 100nucleotides and particularly preferably at least 200 nucleotides.

For efficient translation of the mRNA an productive binding of theribosomes to the ribosome binding site [Kozak sequence: GCCGCCACCAUGG(SEQ ID NO: 13), the AUG forms the start codon] is generally required.In this regard it has been established that an increased A/U contentaround this site facilitates more efficient ribosome binding to themRNA.

In addition, it is possible to introduce one or more so-called IRES(“internal ribosomal entry site”) into the modified mRNA. An IRES mayact as the sole ribosome binding site, or may serve as one of theribosome binding sites of an mRNA. An mRNA comprising more than onefunctional ribosome binding site may encode several peptides orpolypeptides that are translated independently by the ribosomes(“multicistronic mRNA”). Examples of IRES sequences that can be usedaccording to the invention include without limitation, those frompicornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

According to a further preferred embodiment of the present invention themodified mRNA comprises in the 5′ non-translated and/or 3′non-translated regions stabilisation sequences that are capable ofincreasing the half-life of the mRNA in the cytosol.

These stabilisation sequences may exhibit 100% sequence homology withnaturally occurring sequences that are present in viruses, bacteria andeukaryotic cells, or may be derived from such naturally occurringsequences (i.e., may comprise, e.g., mutations substitutions, ordeletions in these sequences). Stabilising sequences that may be used inthe present invention include, by way of non-limiting example, theuntranslated sequences (UTR) of the β-globin gene of Homo sapiens orXenopus laevis. Another example of a stabilisation sequence has thegeneral formula (C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC, which is containedin the 3′UTR of the very stable mRNAs that encode α-globin,α-(I)-collagen, 15-lipoxygenase, or tyrosine hydroxylase (C. F. Holciket al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410-2414). Obviously suchstabilisation sequences may be used individually or in combination, aswell as in combination with other stabilisation sequences known to aperson skilled in the art.

For the further stabilisation of the modified mRNA it is preferred thatthe modified mRNA comprises at least one analogue of a naturallyoccurring nucleotide. This approach is based on the understanding thatRNA-decomposing enzymes present in a cell preferentially recognise RNAcomprising naturally occurring nucleotides as a substrate. The insertionof nucleotide analogues into an RNA molecule, therefore, retardsdecomposition of the RNA molecule so modified, whereas the effect ofsuch analogs on translational efficiency, particularly when insertedinto the coding region of the mRNA, may result in either an increase ordecrease in translation of the modified RNA molecule.

The following is a non-limiting list of nucleotide analogues that can beused in accordance with the invention: phosphorus amidates, phosphorusthioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine,5-methylcytosine and inosine. The preparation of such analogues is knownto the person skilled in the art, for example from U.S. Pat. No.4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat.No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S.Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418,U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530 and U.S. Pat. No.5,700,642. According to the invention such analogues may be present innon-translated and/or translated regions of the modified mRNA.

Furthermore the effective transfer of the modified mRNA into the cellsto be treated or into the organism to be treated may be improved if themodified mRNA is associated with a cationic peptide or protein, or isbound thereto. In particular in this connection the use of protamine aspolycationic, nucleic acid-binding protein is particularly effective. Itis also possible to use other cationic peptides or proteins such aspoly-L-lysine or histones. Procedures for stabilising mRNA are describedin EP-A-1083232, whose relevant disclosure is incorporated herein in itsentirety.

For gene therapy applications, for example, wherein a pharmaceuticalcomposition of the invention is used, the modified mRNA therein codesfor at least one biologically active peptide or polypeptide that is notformed or is only insufficiently or defectively formed in the patient tobe treated. Administration of a modified mRNA encoding the at least onebiologically active peptide or polypeptide or a composition thereof tosuch a patient, therefore, at least partially restores the expressionand/or activity of the at least one biologically active peptide orpolypeptide in the patient and thereby complements the patient's geneticdefect. The direct introduction of a normal, functional gene into aliving animal has been studied as a means for replacing defectivegenetic information. In such studies, nucleic acid sequences areintroduced directly into cells of a living animal. The followingreferences pertain to methods for the direct introduction of nucleicacid sequences into a living animal: Nabel et al., (1990) Science249:1285-1288; Wolfe et al., (1990) Science 247:1465-1468; Acsadi et al.(1991) Nature 352:815-818; Wolfe et al. (1991) BioTechniques11(4):474-485; and Feigner and Rhodes, (1991) Nature 349:351-352, whichare incorporated herein by reference.

Accordingly, examples of polypeptides coded by a modified mRNA of theinvention include, without limitation, dystrophin, the chloride channel,which is defectively altered in cystic fibrosis; enzymes that arelacking or defective in metabolic disorders such as phenylketonuria,galactosaemia, homocystinuria, adenosine deaminase deficiency, etc.;enzymes that are involved in the synthesis of neurotransmitters such asdopamine, norepinephrine and GABA, in particular tyrosine hydroxylaseand DOPA decarboxylase, and α-1-antitrypsin, etc. Pharmaceuticalcompositions of the invention may also be used to effect expression ofcell surface receptors and/or binding partners of cell surface receptorsif the modified mRNA contained therein encodes for such biologicallyactive proteins or peptides. Examples of such proteins that act in anextracellular manner or that bind to cell surface receptors include forexample tissue plasminogen activator (TPA), growth hormones, insulin,interferons, granulocyte-macrophage colony stimulating factor (GM-CFS),and erythropoietin (EPO), etc. By choosing suitable growth factors, thepharmaceutical composition of the present invention may, for example, beused for tissue regeneration. In this way diseases that arecharacterised by tissue degeneration, for example neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, etc. andother degenerative conditions, such as arthrosis, can be treated. Inthese cases the modified mRNA, in particular that contained in thepharmaceutical composition of the present invention, preferably encodes,without limitation, a TGF-β family member, EGF, FGF, PDGF, BMP, GDNF,BDNF, GDF and neurotrophic factors such as NGF, neutrophines, etc.

A further area of application of the present invention is vaccination,i.e. the use of a modified mRNA for inoculation or the use of apharmaceutical composition comprising a modified mRNA as an inoculatingagent, or the use of a modified mRNA in the preparation of thepharmaceutical composition for inoculation purposes. Vaccination isbased on introducing an antigen into an organism or subject, inparticular into a cell of the organism or subject. In the context of thepresent invention, the genetic information encoding the antigen isintroduced into the organism or subject in the form of a modified mRNAencoding the antigen. The modified mRNA contained in the pharmaceuticalcomposition is translated into the antigen, i.e. the polypeptide orantigenic peptide coded by the modified mRNA is expressed, and an immuneresponse directed against the polypeptide or antigenic peptide isstimulated. For vaccination against a pathogenic organism, e.g., avirus, a bacterium, or a protozoan, a surface antigen of such anorganism maybe used as an antigen against which an immune response iselicited. In the context of the present invention, a pharmaceuticalcomposition comprising a modified mRNA encoding such a surface antigenmay be used as a vaccine. In applications wherein a genetic vaccine isused for treating cancer, the immune response is directed against tumourantigens by generating a modified mRNA encoding a tumour antigen(s), inparticular a protein which is expressed exclusively on cancer cells.Such a modified mRNA encoding a tumour antigen may be used alone or as acomponent of a pharmaceutical composition according to the invention,wherein administration of either the modified mRNA or a compositionthereof results in expression of the cancer antigen(s) in the organism.An immune response to such a vaccine would, therefore, confer to thevaccinated subject a degree of protective immunity against cancersassociated with the immunizing cancer antigen. Alternatively, suchmeasures could be used to vaccinate a cancer patient with a modifiedmRNA encoding a tumour antigen(s) expressed on the patient's cancercells so as to stimulate the cancer patient's immune response to attackany cancer cells expressing the encoded antigen.

In its use as a vaccine the pharmaceutical composition according to theinvention is suitable in particular for the treatment of cancers (inwhich the modified mRNA codes for a tumour-specific surface antigen(TSSA), for example for treating malignant melanoma, colon carcinoma,lymphomas, sarcomas, small-cell lung carcinomas, blastomas, etc. Anon-limiting list of specific examples of tumour antigens include, interalia, 707-AP, AFP, ART-4, BAGE, β-catenin/m, Bcr-abl, CAMEL, CAP-1,CASP-8, CDC27/m, CDK4/m, CEA, CT, Cyp-β, DAM, ELF2M, ETV6-AML1, G250,GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M,HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR/FUT, MAGE,MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1,p190 minor bcr-abl, Pml/RARα, PRAME, PSA, PSM, RAGE, RU1 or RU2, SAGE,SART-1 or SART-3, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 and WT1. Inaddition to the above application, the pharmaceutical composition of theinvention may be used to treat infectious diseases, for example, viralinfectious diseases such as AIDS (HIV), hepatitis A, B or C, herpes,herpes zoster (chicken pox), German measles (rubella virus), yellowfever, dengue fever etc. (flavi viruses), flu (influenza viruses),haemorrhagic infectious diseases (Marburg or Ebola viruses), bacterialinfectious diseases such as Legionnaires' disease (Legionella), gastriculcer (Helicobacter), cholera (Vibrio), E. coli infections,staphylococcal infections, salmonella infections or streptococcalinfections, tetanus (Clostridium tetani), or protozoan infectiousdiseases (malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e.infections caused by plasmodium, trypanosomes, leishmania andtoxoplasma). Preferably also in the case of infectious diseases thecorresponding surface antigens with the strongest antigenic potentialare encoded by the modified mRNA. With the aforementioned genes ofpathogenic vectors or organisms, in particular in the case of viralgenes, this is typically a secreted form of a surface antigen. Moreover,according to the invention mRNAs preferably coding for polypeptides areemployed, because polypeptides generally comprise multiple epitopes(polyepitopes). Polypeptides comprising polyepitopes include but are notlimited to, surface antigens of pathogenic vectors or organisms, or oftumour cells, preferably secreted protein forms.

Moreover, the modified mRNA according to the invention may comprise inaddition to the antigenic or therapeutically active peptide orpolypeptide, at least one further functional region that encodes, forexample, a cytokine that promotes the immune response (e.g., a monokine,lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, GM-CFS, LT-αor growth factors such as hGH).

Furthermore, in order to increase immunogenicity, the pharmaceuticalcomposition according to the invention may contain one or moreadjuvants. The term “adjuvant” is understood in this context to denoteany chemical or biological compound that promotes or augments a specificimmune response. Various mechanisms may be involved in this connection,depending on the various types of adjuvants. For example, compounds thatpromote endocytosis of the modified mRNA contained in the pharmaceuticalcomposition by dentritic cells (DC) form a first class of usableadjuvants. Other compounds that activate or accelerate maturation of DC(for example, lipopolysaccharides, TNF-α or CD40 ligand) comprise asecond class of suitable adjuvants. In general, any agent which isrecognized as a potential “danger signal” by the immune system (LPS,GP96, oligonucleotides with the CpG motif) or cytokines such as GM-CSF,may be used as an adjuvant. Co-administration of an adjuvant enhances animmune response generated against an antigen encoded by the modifiedmRNA. The aforementioned cytokines are particularly preferred in thisaspect. Other known adjuvants include aluminium hydroxide, and Freund'sadjuvant, as well as the aforementioned stabilising cationic peptides orpolypeptides such as protamine. In addition, lipopeptides such asPam3Cys are also particularly suitable for use as adjuvants in thepharmaceutical composition of the present invention; see Deres et al,Nature 1989, 342: 561-564.

The pharmaceutical composition according to the invention comprises, inaddition to the modified mRNA, a pharmaceutically compatible carrierand/or a pharmaceutically compatible vehicle. Appropriate methods forachieving a suitable formulation and preparation of the pharmaceuticalcomposition according to the invention are described in “Remington'sPharmaceutical Sciences” (Mack Pub. Co., Easton, Pa., 1980), which isherein incorporated by reference in its entirety. For parenteraladministration suitable carriers include for example sterile water,sterile saline solutions, polyalkylene glycols, hydrogenated naphthaleneand in particular biocompatible lactide polymers, lactide/glycolidecopolymers or polyoxyethylene/polyoxypropylene copolymers. Compositionsaccording to the invention may contain fillers or substances such aslactose, mannitol, substances for the covalent coupling of polymers suchas for example polyethylene glycol to inhibitors according to theinvention, complexing with metal ions or incorporation of materials inor on special preparations of polymer compound, such as for examplepolylactate, polyglycolic acid, hydrogel or on liposomes,microemulsions, microcells, unilamellar or multilamellar vesicles,erythrocyte fragments or spheroplasts. The respective modifications ofthe compositions are chosen depending on physical properties such as,for example, solubility, stability, bioavailability or degradability.Controlled or constant release of the active component according to theinvention in the composition includes formulations based on lipophilicdepot substances (for example fatty acids, waxes or oils). Coatings ofsubstances or compositions according to the invention containing suchsubstances, namely coatings with polymers (for example poloxamers orpoloxamines), are also disclosed within the scope of the presentinvention. Moreover substances or compositions according to theinvention may contain protective coatings, for example proteaseinhibitors or permeability enhancers. Preferred carriers are typicallyaqueous carrier materials, in which water for injection (WFI) or waterbuffered with phosphate, citrate or acetate, etc., is used, and the pHis typically adjusted to 5.0 to 8.0, preferably 6.0 to 7.0. The carrieror the vehicle will in addition preferably contain salt constituents,for example sodium chloride, potassium chloride or other components thatfor example make the solution isotonic. In addition the carrier or thevehicle may contain, besides the aforementioned constituents, additionalcomponents such as human serum albumin (HSA), polysorbate 80, sugars oramino acids.

The concentration of the modified mRNA in such formulations maytherefore vary within a wide range from 1 μg to 100 mg/ml. Thepharmaceutical composition according to the invention is preferablyadministered parenterally, for example intravenously, intraarterially,subcutaneously or intramuscularly to the patient. It is also possible toadminister the pharmaceutical composition topically or orally.

The invention thus also provides a method for the treatment of theaforementioned medical conditions or an inoculation method for theprevention of the aforementioned conditions, which comprises theadministration of the pharmaceutical composition according to theinvention to a subject or patient, in particular a human patient.

A typical regimen for preventing, suppressing, or treating a pathologyrelated to a viral, bacterial, or protozoan infection, may compriseadministration of an effective amount of a vaccine composition asdescribed herein, administered as a single treatment, or repeated asenhancing or booster dosages, over a period up to and including betweenone week and about 24 months, or any range or value therein.

According to the present invention, an “effective amount” of a vaccinecomposition is one that is sufficient to achieve a desired biologicaleffect. It is understood that nature and manner of the administrationand the effective dosage may be determined by a medical practitionerbased on a number of variables including the age, sex, health, andweight of the recipient, the medical condition to be treated and itsstage of progression, the kind of concurrent treatment, if any,frequency of treatment, and the nature of the desired outcome. Theranges of effective doses provided below are not intended to limit theinvention, but are provided as representative preferred dose ranges.However, the most preferred dosage will be tailored to the individualsubject, as is understood and determinable by one of skill in the art,without undue experimentation. See, e.g., Berkow et al., eds., The MerckManual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman et al.,eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics,8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co.,Boston, Mass. (1985); and Katzung, ed. Basic and Clinical Pharmacology,Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992), whichreferences and references cited therein, are entirely incorporatedherein by reference.

The present invention relates to the use of genetic material (e.g.,nucleic acid sequences) as immunizing agents. In one aspect, the presentinvention relates to the introduction of exogenous or foreign modifiedDNA or RNA molecules into an individual's tissues or cells, whereinthese molecules encode an exogenous protein capable of eliciting animmune response to the protein. The exogenous nucleic acid sequences maybe introduced alone or in the context of an expression vector whereinthe sequences are operably linked to promoters and/or enhancers capableof regulating the expression of the encoded proteins. The introductionof exogenous nucleic acid sequences may be performed in the presence ofa cell stimulating agent capable of enhancing the uptake orincorporation of the nucleic acid sequences into a cell. Such exogenousnucleic acid sequences may be administered in a composition comprising abiologically compatible or pharmaceutically acceptable carrier. Theexogenous nucleic acid sequences may be administered by a variety ofmeans, as described herein, and well known in the art.

Such methods may be used to elicit immunity to a pathogen, absent therisk of infecting an individual with the pathogen. The present inventionmay be practiced using procedures known in the art, such as thosedescribed in PCT International Application Number PCT/US90/01515,wherein methods for immunizing an individual against pathogen infectionby directly injecting polynucleotides into the individual's cells in asingle step procedure are presented.

In one aspect, the present invention relates to methods for elicitingimmune responses in an individual or subject which can protect theindividual from pathogen infection. Accordingly, genetic material thatencodes an immunogenic protein is introduced into a subject's cellseither in vivo or ex vivo. The genetic material is expressed by thesecells, thereby producing immunogenic target proteins capable ofeliciting an immune response. The resulting immune response is broadbased and involves activation of the humoral immune response and botharms of the cellular immune response.

This approach is useful for eliciting a broad range of immune responsesagainst a target protein. Target proteins may be proteins specificallyassociated with pathogens or the individual's own “abnormal” or infectedcells. Such an approach may be used advantageously to immunize a subjectagainst pathogenic agents and organisms such that an immune responseagainst a pathogen protein provides protective immunity against thepathogen. This approach is particularly useful for protecting anindividual against infection by non-encapsulated intracellularpathogens, such as a virus, which produce proteins within the hostcells. The immune response generated against such proteins is capable ofeliminating infected cells with cytotoxic T cells (CTLs).

The immune response elicited by a target protein produced by vaccinatedcells in a subject is a broad-based immune response which includes Bcell and T cell responses, including CTL responses. It has been observedthat target antigen produced within the cells of the host are processedintracellularly into small peptides, which are bound by Class I MHCmolecules and presented in the context of Class I on the cell surface.The Class I MHC-target antigen complexes are capable of stimulating CD8⁺T cells, which are predominantly CTLs. Notably, genetic immunizationaccording to the present invention is capable of eliciting CTL responses(killer cell responses).

The CTL response is crucial in protection against pathogens such asviruses and other intracellular pathogens which produce proteins withininfected cells. Similarly, the CTL response can be utilized for thespecific elimination of deleterious cell types, which may expressaberrant cell surface proteins recognizable by Class I MHC molecules.

The genetic vaccines of the present invention may be administered tocells in conjunction with compounds that stimulate cell division andfacilitate uptake of genetic constructs. This step provides an improvedmethod of direct uptake of genetic material. Administration of cellstimulating compounds results in a more effective immune responseagainst the target protein encoded by the genetic construct.

According to the present invention, modified DNA or mRNA that encodes atarget protein is introduced into the cells of an individual where it isexpressed, thus producing the target protein. The modified DNA or RNAmay be operably linked to regulatory elements (e.g., a promoter)necessary for expression in the cells of the individual. Other elementsknown to skilled artisans may also be included in genetic constructs ofthe invention, depending on the application.

As used herein, the term “genetic construct” refers to the modified DNAor mRNA molecule that comprises a nucleotide sequence which encodes thetarget protein and which may include initiation and termination signalsoperably linked to regulatory elements including a promoter andpolyadenylation signal (for modified DNA) capable of directingexpression in the cells of the vaccinated individual. As used herein,the term “expressible form” refers to gene constructs which contain thenecessary regulatory elements operably linked to a coding sequence of atarget protein, such that when present in the cell of the individual,the coding sequence is expressed. As used herein, the term “geneticvaccine” refers to a pharmaceutical preparation that comprises a geneticconstruct.

The present invention provides genetic vaccines, which include geneticconstructs comprising DNA or RNA which encode a target protein. As usedherein, the term “target protein” refers to a protein capable ofeliciting an immune response. The target protein is an immunogenicprotein derived from the pathogen or undesirable cell-type, such as aninfected or transformed cell. In accordance with the invention, targetproteins may be pathogen-associated proteins or tumour-associatedproteins. The immune response directed against the target proteinprotects the individual against the specific infection or disease withwhich the target protein is associated. For example, a genetic vaccinecomprising a modified DNA or RNA molecule that encodes apathogen-associated target protein is used to elicit an immune responsethat will protect the individual from infection by the pathogen.

DNA and RNA-based vaccines and methods of use are described in detail inseveral publications, including Leitner et al. (1999, Vaccines18:765-777), Nagashunmugam et al. (1997, AIDS 11:1433-1444), and Fleetonet al. (2001, J Infect Dis 183:1395-1398) the entire contents of each ofwhich is incorporated herein by reference.

In order to test expression, genetic constructs can be tested forexpression levels in vitro using cells maintained in culture, which areof the same type as those to be vaccinated. For example, if the geneticvaccine is to be administered into human muscle cells, muscle cellsgrown in culture such as solid muscle tumor cells of rhabdomyosarcomamay be used as an in vitro model for measuring expression levels. One ofordinary skill in the art could readily identify a model in vitro systemwhich may be used to measure expression levels of an encoded targetprotein.

In accordance with the invention, multiple inoculants can be deliveredto different cells, cell types, or tissues in an individual. Suchinoculants may comprise the same or different nucleic acid sequences ofa pathogenic organism. This allows for the introduction of more than asingle antigen target and maximizes the chances for developing immunityto the pathogen in a vaccinated subject.

According to the invention, the genetic vaccine may be introduced invivo into cells of an individual to be immunized or ex vivo into cellsof the individual which are re-implanted after incorporation of thegenetic vaccine. Either route may be used to introduce genetic materialinto cells of an individual. As described herein above, preferred routesof administration include intramuscular, intraperitoneal, intradermal,and subcutaneous injection. Alternatively, the genetic vaccine may beintroduced by various means into cells isolated from an individual. Suchmeans include, for example, transfection, electroporation, andmicroprojectile bombardment. These methods and other protocols forintroducing nucleic acid sequences into cells are known to and routinelypracticed by skilled practitioners. After the genetic construct isincorporated into the cells, they are re-implanted into the individual.Prior to re-implantation, the expression levels of a target proteinencoded by the genetic vaccine may be assessed. It is contemplated thatotherwise non-immunogenic cells that have genetic constructsincorporated therein can be implanted into autologous or heterologousrecipients.

The genetic vaccines according to the present invention comprise about0.1 to about 1000 micrograms of nucleic acid sequences (i.e., DNA orRNA). In some preferred embodiments, the vaccines comprise about 1 toabout 500 micrograms of nucleic acid sequences. In some preferredembodiments, the vaccines comprise about 25 to about 250 micrograms ofnucleic acid sequences. Most preferably, the vaccines comprise about 100micrograms nucleic acid sequences.

The genetic vaccines according to the present invention are formulatedaccording to the mode of administration to be used. One having ordinaryskill in the art can readily formulate a genetic vaccine that comprisesa genetic construct. In cases where intramuscular injection is thechosen mode of administration, for example, an isotonic formulation isgenerally used. As described in detail herein above, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. Isotonic solutions such as phosphate buffered saline arepreferred. Stabilizers can include gelatin and albumin.

In some embodiments of the invention, the individual is administered aseries of vaccinations to produce a comprehensive immune response.According to this method, at least two and preferably four injectionsare given over a period of time. The period of time between injectionsmay include from 24 hours apart to two weeks or longer betweeninjections, preferably one week apart. Alternatively, at least two andup to four separate injections may be administered simultaneously todifferent parts of the body.

While this disclosure generally discusses immunization or vaccination inthe context of prophylactic methods of protection, the terms“immunizing” or “vaccinating” are meant to refer to both prophylacticand therapeutic methods. Thus, a method for immunizing or vaccinatingincludes both methods of protecting an individual from pathogenchallenge, as well as methods for treating an individual suffering frompathogen infection. Accordingly, the present invention may be used as avaccine for prophylactic protection or in a therapeutic manner; that is,as a reagent for immunotherapeutic methods and preparations.

The amount of a modified nucleic acid sequence generated using themethods of the invention which provides a therapeutically effective dosein the treatment of a patient with, for example, cancer or apathogen-related disorder can be determined by standard clinicaltechniques based on the present description. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each subject's circumstances. However, suitabledosage ranges for intravenous administration are generally directed toachieve a concentration of about 20-500 micrograms of polypeptideencoded by the modified nucleic acid per kilogram body weight. Suitabledosage ranges for intranasal administration are generally directed toachieve a concentration of about 0.01 pg to 1 mg of polypeptide encodedby the modified nucleic acid per kg body weight. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

The compositions comprising the modified nucleic acid molecules of theinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic applications, compositions are administeredto a patient already suffering from a hyperproliferative disorder (suchas, e.g., cancer) in an amount sufficient to cure or at least partiallyarrest the symptoms of the disease and its complications. An amountadequate to accomplish this is defined as a “therapeutically effectiveamount or dose.” Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient.

Compositions comprising modified nucleic acid molecules of the inventioncan be administered alone, or in combination, and/or in conjunction withknown therapeutic agents/compounds used for the treatment of a patientwith a particular disorder. For the treatment of a patient with cancer,for example, a composition comprising at least one modified nucleic acidof the invention which encodes a tumour antigen, may be used inconjunction with one or more known cancer therapeutics, such as thosedescribed in the Physicians' Desk Reference, 54^(th) Edition (2000) orin Cancer: Principles & Practice of Oncology, DeVita, Jr., Hellman, andRosenberg (eds.) 2nd edition, Philadelphia, Pa.: J.B. Lippincott Co.,1985, wherein standard treatment protocols and dosage formulations arepresented.

In addition a method is also provided for determining how to modify thesequence of an mRNA so as to generate a modified mRNA having alteredproperties, which may be used alone or in a pharmaceutical compositionof the invention. In this connection, and in accordance with theinvention, the modification of an RNA sequence is carried out with twodifferent optimisation objectives: to maximize G/C content, and tomaximize the frequency of codons that are recognized by abundantlyexpressed tRNAs. In the first step of the process a virtual translationof an arbitrary RNA (or DNA) sequence is carried out in order togenerate the corresponding amino acid sequence. Starting from the aminoacid sequence, a virtual reverse translation is performed that provides,based on degeneracy of the genetic code, all of the possible choices forthe corresponding codons. Depending on the required optimisation ormodification, corresponding selection lists and optimisation algorithmsare used for choosing suitable codons. The algorithms are executed on acomputer, normally with the aid of suitable software. In accordance withthe present invention, a suitable software program comprises a sourcecode of Appendix I. Thus, the optimised mRNA sequence is generated andcan be output, for example, with the aid of a suitable display deviceand compared with the original (wild type) sequence. The same alsoapplies with regard to the frequency of the individual nucleotides. Thechanges compared to the original nucleotide sequence are preferablyemphasised. Furthermore, according to a preferred embodiment, naturallyoccurring stable sequences are incorporated therein to produce an RNAstabilised by the presence of natural sequence motifs. A secondarystructural analysis may also be performed that can analyse, on the basisof structural calculations, stabilising and destabilising properties orregions of the RNA.

Also encompassed by the present invention are modified nucleic acidsequences generated using the above computer-based method. Exemplarymodified nucleic acid sequences of the invention include SEQ ID NOs:3-7, 10 and 11. The present invention also includes pharmaceuticalcompositions of modified nucleic acid sequences of the invention,including SEQ ID NOs: 3-7, 10 and 11.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G show wild type sequences and modified sequences for theinfluenza matrix protein.

FIG. 1A (SEQ ID NO: 1) shows the wild type gene and FIG. 1B (SEQ ID NO:2) shows the amino acid sequence derived therefrom (1-letter code). FIG.1C (SEQ ID NO: 3) shows a gene sequence coding for the influenza matrixprotein, whose G/C content is increased as compared to that of the wildtype sequence. FIG. 1D (SEQ ID NO: 4) shows the sequence of a gene thatcodes for a secreted form of the influenza matrix protein (including anN-terminal signal sequence), wherein the G/C content of the sequence isincreased relative to that of the wild type sequence. FIG. 1E (SEQ IDNO: 5) shows an mRNA coding for the influenza matrix protein, whereinthe mRNA comprises stabilising sequences not present in thecorresponding wild type mRNA. FIG. 1F (SEQ ID NO: 6) shows an mRNAcoding for the influenza matrix protein that in addition to stabilisingsequences also contains an increased G/C content. FIG. 1G (SEQ ID NO: 7)likewise shows a modified mRNA that codes for a secreted form of theinfluenza matrix protein and comprises, as compared to the wild type,stabilising sequences and an elevated G/C content. In FIG. 1A and FIGS.1C to 1G the start and stop codons are shown in bold type. Nucleotidesthat are changed relative to the wild type sequence of FIG. 1A are shownin capital letters in 1C to 1G.

FIGS. 2A-D show wild type sequences and modified sequences according tothe invention that encode for the tumour antigen MAGE1.

FIG. 2A (SEQ ID NO: 8) shows the sequence of the wild type gene and FIG.2B (SEQ ID NO: 9) shows the amino acid sequence derived therefrom(3-letter code). FIG. 2C (SEQ ID NO: 10) shows a modified mRNA codingfor MAGE1, whose G/C content is increased as compared to the wild type.FIG. 2D (SEQ ID NO: 11) shows the sequence of a modified mRNA encodingMAGE1, in which the codon usage has been optimised as frequently aspossible with respect to the tRNA present in the cell and to the codingsequence in question. Start and stop codons are shown in each case inbold type.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples describe the invention in more detail and in noway are to be construed as restricting the scope thereof.

Example 1

As an exemplary embodiment of the process for determining the sequenceof a modified mRNA according to the invention, a computer program wasestablished that modifies the nucleotide sequence of an arbitrary mRNAin such a way as to maximise the G/C content of the nucleic acid, andmaximise the presence of codons recognized by abundant tRNAs present ina particular cell(s). The computer program is based on an understandingof the genetic code and exploits the degenerative nature of the geneticcode. By this means a modified mRNA having desirable properties isobtained, wherein the amino acid sequence encoded by the modified mRNAis identical to that of the unmodified mRNA sequence. Alternatively, theinvention may encompass alterations in either the G/C content or codonusage of an mRNA to produce a modified mRNA.

The source code in Visual Basic 6.0 (program development environmentemployed: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) isgiven in the Appendix I.

Example 2

An RNA construct with a sequence of the lac-Z gene from E. colioptimised with regard to stabilisation and translational efficiency wasproduced with the aid of the computer program of Example 1. A G/Ccontent of 69% (compared to the wild type sequence of 51%; C. F. Kalninset al., EMBO J. 1983, 2(4): 593-597) was achieved in this manner.Through the synthesis of overlapping oligonucleotides that comprise themodified sequence, the optimised sequence was produced according tomethods known in the art. The terminal oligonucleotides have thefollowing restriction cleavage sites: at the 5′ end an EcoRV cleavagesite, and at the 3′ end a BglII cleavage site. The modified lacZsequence was incorporated into the plasmid pT7 Ts (GenBank Accession No.U26404; C. F. Lai et al., see above) by digestion with EcoRV/BglII. pT7Ts contains untranslated region sequences from the β-globin gene ofXenopus laevis at the 5′ and 3′ ends. The plasmid was cleaved with theaforementioned restriction enzymes to facilitate insertion of themodified lacZ sequence having compatible 5′ and 3′ termini.

The pT7 Ts-lac-Z construct was propagated in bacteria and purified byphenol-chloroform extraction. 2 μg of the construct were transcribed invitro using methods known to a skilled artisan and the modified mRNA wasproduced.

Example 3

The gene for the influenza matrix protein (wild type sequence, see FIG.1A; derived amino acid sequence, see FIG. 1B) was optimised with the aidof the computer program according to the invention of Example 1. TheG/C-rich sequence variant shown in FIG. 1C (SEQ ID NO: 3) was therebyformed. A G/C-rich sequence coding for a secreted form of the influenzamatrix protein, which includes an N-terminal signal sequence was alsodetermined (see FIG. 1D; SEQ ID NO: 4). The secreted form of theinfluenza matrix protein has the advantage of increased immunogenicityas compared to that of the non-secreted form.

Corresponding mRNA molecules were designed starting from the optimisedsequences. The mRNA for the influenza matrix protein, optimised withregard to G/C content and codon usage, was additionally provided withstabilising sequences in the 5′ region and 3′ region (the stabilisationsequences derive from the 5′-UTRs and 3′-UTRs of the β-globin-mRNA ofXenopus laevis; pT7 Ts-Vektor in C. F. Lai et al., see above). See alsoFIG. 1E; SEQ ID NO: 5, which includes only stabilising sequences and 1F;SEQ ID NO: 6, which includes both increased G/C content and stabilisingsequences. The mRNA coding for the secreted form of the influenza matrixprotein was likewise also sequence optimised in the translated regionand provided with the aforementioned stabilising sequences (see FIG. 1G;SEQ ID NO: 7).

Example 4

The mRNA encoding the tumour antigen MAGE1 was modified with the aid ofthe computer program of Example 1. The sequence shown in FIG. 2C (SEQ IDNO: 10) was generated in this way, and has a 24% higher G/C content (351G, 291 C) as compared to the wild type sequence (275 G, 244 G). Inaddition, by means of alternative codon usage, the wild type sequencewas improved with regard to translational efficiency by substitutingcodons corresponding to tRNAs that are more abundant in a target cell(see FIG. 2D; SEQ ID NO: 11). The G/C content was likewise raised by 24%by the alternative codon usage.

What is claimed is:
 1. A method of providing insulin expression in asubject comprising administering an effective amount of a pharmaceuticalcomposition comprising a mRNA that encodes insulin to the subject,wherein the mRNA that encodes insulin comprises: (i) an increased G/Ccontent relative to a wild type RNA encoding for insulin; or (ii) atleast one codon of a wild-type sequence recognized by a rare cellulartRNA is replaced with a codon recognized by an abundant cellular tRNA,and wherein said rare cellular tRNA and said abundant cellular tRNArecognize the same amino acid.
 2. The method of claim 1, wherein thepharmaceutical composition is administered by injection.
 3. The methodof claim 1, wherein the pharmaceutical composition is administeredintravenously, intradermally, subcutaneously, intramuscularly, topicallyor orally.
 4. The method of claim 3, wherein the pharmaceuticalcomposition is administered intravenously.
 5. The method of claim 1,wherein the mRNA that encodes insulin comprises an increased G/C contentrelative to a wild type RNA encoding for insulin.
 6. The method of claim1, wherein the mRNA encoding insulin comprises a sequence wherein atleast one codon of a wild-type sequence recognized by a rare cellulartRNA is replaced with a codon recognized by an abundant cellular tRNA,and wherein said rare cellular tRNA and said abundant cellular tRNArecognize the same amino acid.
 7. The method of claim 1, wherein themRNA encoding insulin comprises at least one chemical modification ofthe mRNA.
 8. The method of claim 1, wherein the mRNA encoding insulincomprises at least one nucleotide of the mRNA is substituted with ananalog of the naturally occurring nucleotide.
 9. The method of claim 1,wherein the mRNA encoding insulin comprises at least one nucleotideposition replaced with a nucleotide analogue selected from the groupconsisting of phosphorus amidates, phosphorus thioates, peptidenucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine andinosine.
 10. The method of claim 1, wherein the mRNA encoding insulincomprises a stabilizing 5′ untranslated region (UTR) or 3′ UTR.
 11. Themethod of claim 1, wherein the mRNA comprises a 5′ cap structure. 12.The method of claim 1, wherein the mRNA comprises a poly-A tail of atleast 50 nucleotides.
 13. The method of claim 1, wherein the mRNA isdissolved in the aqueous carrier.
 14. The method of claim 13, whereinthe aqueous carrier is water for injection (WFI), a buffered solution ora salt solution.
 15. The method of claim 14, wherein the salt solutioncomprises sodium chloride or potassium chloride solution.
 16. The methodof claim 1, wherein the pharmaceutical composition comprises a componentselected from the group consisting of human serum albumin, apolycationic protein, polysorbate 80, a sugar and an amino acid.
 17. Themethod of claim 16, wherein the pharmaceutical composition comprises apolycationic protein.
 18. The method of claim 1, wherein the mRNA isprovided in a liposome complex.
 19. The method of claim 1, furthercomprising administering the pharmaceutical composition to the subjecttwo or more times.
 20. The method of claim 1, wherein the composition isadministered by injection and wherein the mRNA is provided in a liposomecomplex.
 21. The method of claim 1, wherein the subject has insufficientlevels of insulin.